DESCRIPTION: This revised application is concerned with the development of an in vitro expression technology (IVET) model for Actinobacillus actinomycetemcomitans (A.a.). The hypothesis is that genes encoding virulence factors in A. a. can be selected in vivo using the appropriate technology. The experimental plan is to develop a plasmid vector with markers which can be selected in E. coli and A.a. A genomic library of A.a. will be constructed in E. coli using the novel vector. Subsequently, the library will be purified and used to transform A.a such that the plasmid-borne DNA will integrate into the chromosome creating merodiploid strains of A.a. The merodiploid strains of A.a. will then be introduced in a mouse using a peritoneal abscess model. Bacteria surviving antibiotic selection will then be recovered from peripheral blood samples and examined for their genetic structure. After the construction of a heterodiploid library of strains, the longer-range goal of the project is to use the strains in a murine abscess model to select strains which are expressing virulence traits. Such strains will be identified following the plating of blood samples from infected animals. Support is sought in this proposal for development of the appropriate vector and its analysis, using a known promoter for the A.a. leukotoxin gene, in an animal model.